Investigating the individual and mixture cytotoxicity of co-occurring aflatoxin B1, enniatin B, and sterigmatocystin on gastric, intestinal, hepatic, and renal cellular models.

This study investigates the individual and combined effects of the mycotoxins, Aflatoxin B1 (AFB1), Enniatin B (ENNB) and Sterigmatocystin (STG), on the cellular viability of gastric (NCI-N87), intestinal (Caco-2), hepatic (Hep-G2) and renal (Hek-293) cells, shedding light on synergistic or antagonistic effects using a constant ratio combination design proposed by Chou-Talalay. These toxins are prevalent in cereal-based foods, frequently consumed by children which raises concerns about their exposure to these mycotoxins. This population is particularly vulnerable to the effects of these toxins due to their underdeveloped organs and incompletely structured physiological processes. Results showed that ENB was the most toxic of the three mycotoxins across all cell lines, while STG and AFB1 showed lower toxicity. The combination of ENNB + STG was found to be the most potent in terms of binary mixtures. In regard to ternary combinations, Caco-2 cells are more sensitive to the tested mycotoxins, whereas NCI-N87 cells show lower levels of cell damage. Worrying dose reduction values (>10-fold) were found for ENNB in binary and ternary combinations at low exposure levels. These findings are significant for establishing initial reference values, which play a pivotal role in estimating reference doses that are subsequently incorporated into the broader risk assessment process.

Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method.

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.

Development of an in vitro neuroblastoma 3D model and its application for sterigmatocystin-induced cytotoxicity testing.

Given the increasing importance of establishing better risk assessments for mycotoxins, novel in vitro tools for the evaluation of their toxicity are mandatory. In this study, an in vitro 3D spheroid model from SH-SY5Y cells, a human neuroblastoma cell line, was developed, optimized and characterized to test the cytotoxic effects caused by the mycotoxin sterigmatocystin (STE). STE induced a concentration- and time-dependent cell viability decrease in spheroids. Spheroids displayed cell disaggregation after STE exposure, increasing in a dose-dependent manner and over time. STE also induced apoptosis as confirmed by immunofluorescence staining and Western blot. Following the decreased proliferation and increased apoptosis, STE cytostasis effects were observed by migration assays both in 2D and 3D cell culture. Increased ROS generation, as well as DNA damage were also observed. Taken together, these data highlight the cytotoxic properties of STE and suggest that cell culture models play a pivotal role in the toxicological risk assessment of mycotoxins. The evaluation of cytotoxicity in spheroids (3D) rather than monolayer cultures (2D) is expected to more accurately reflect in vivo-like cell behaviour.

Role of quercetin on sterigmatocystin-induced oxidative stress-mediated toxicity.

Oxidative stress appears to be a common trigger for many of the effects associated with the exposure to various mycotoxins, including sterigmatocystin (STE). However, studies to alleviate STE toxicity through the use of natural antioxidants are sparsely reported in literature. In the present study, the cytoprotective effect of quercetin (QUE) was tested in SH-SY5Y cells against STE-induced oxidative stress and cytotoxicity. The MTT assay revealed that STE decreased cell viability, whereas pre-treatment of cells with QUE restored it. The QUE was also found to counteract STE-induced ROS generation and decrease STE-induced up-regulation of the expression of the stress-inducible enzymes HO-1 and NOS-2. Pre-treatment with QUE also prevented STE-induced nuclear translocation of NF-κB, as measured by immunofluorescence. Finally, considering the key role played by NF-κB in the regulation of inflammation, the effect of STE on the pro-inflammatory cytokines TNF-α and IL-6 expression was evaluated. Our results showed the down-regulation of TNF-α and IL-6 following STE exposure, suggesting a negative immunomodulatory effect of STE. In QUE pre-treated samples, TNF-α and IL-6 were significantly further reduced, indicating the anti-inflammatory role of QUE. The results of the present study demonstrate for the first time that QUE exerts a cytoprotective role in STE-induced toxicity.

Sterigmatocystin, 5-Methoxysterigmatocistin, and Their Combinations Are Cytotoxic and Genotoxic to A549 and Hepg2 Cells and Provoke Phosphorylation of Chk2, but Not Fancd2 Checkpoint Proteins.

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are structurally related mycotoxins with cytotoxic and genotoxic properties. In the present study, we hypothesized that DNA damage induced by non-cytotoxic concentrations of single and combined mycotoxins could alter the phosphorylation of the checkpoint proteins Chk2 and FANCD2 (ELISA) in HepG2 and A549 cells. The cytotoxic potential (MTT test) of single and combined STC and 5-M-STC, the nature of their interaction (additivity, antagonism, or synergy) and DNA damage level (alkaline comet assay) in HepG2 and A549 cells were also investigated. All experiments were performed after 24 h of mycotoxin treatment. 5-M-STC was 10-folds more cytotoxic than STC to both HepG2 and A549 cells. Both mycotoxins are genotoxic to HepG2 and A549 cells by inducing both double and single DNA strand breaks that activate Chk2 (especially in HepG2 cells) but not the FANCD2 protein. STC exerted higher genotoxic potential than 5-M-STC in HepG2 and A549 cells when both toxins were applied individually at the same concentration. Dual combinations of non-cytotoxic mycotoxin concentrations showed additive to antagonizing cytotoxic and genotoxic effects. The absence and low activation of checkpoint proteins during prolonged exposure to non-cytotoxic concentrations of STC and 5-M-STC could support cell proliferation and carcinogenesis.

Sterigmatocystin-induced DNA damage triggers cell-cycle arrest via MAPK in human neuroblastoma cells.

Sterigmatocystin (STE) is a common mycotoxin found in food and feed. Many studies showed that STE is genotoxic. However, up to now, the potential genotoxicity of STE on human neuronal system remains unknown. In this study, we explored the effect of STE on DNA damage and cell-cycle progression on human neuroblastoma SH-SY5Y cells exposed to various concentrations of STE (0.78, 1.56 and 3.12 µM) for 24 h. The results indicated that STE exposure induced DNA damage, as evidenced by DNA comet tails formation and increased γH2AX foci. Additionally, genotoxicity was confirmed by micronuclei (MN) analysis. Furthermore, we found that STE exposure led to cell-cycle arrest at the S and the G2/M phase. Considering the important role played by MAPK and p53 signaling pathways in cell-cycle arrest, we explored their potential involvement in STE-induced cell-cycle arrest by using specific inhibitors. The inhibition of JNK and ERK resulted to attenuate S and G2/M arrest, whereas the inhibition of p38 and p53 attenuated only STE-induced S phase arrest. In conclusion, the present study demonstrates that STE induced DNA damage and triggered MAPK and p53 pathways activation, resulting in cell-cycle arrest at the S and the G2/M phase.

Individual and Combined Effects of Aflatoxin B1 and Sterigmatocystin on Lipid Peroxidation and Glutathione Redox System of Common Carp Liver.

The purpose of the study was to evaluate the short-term effects of aflatoxin B1 (AFB1 100 µg/kg feed) and sterigmatocystin (STC 1000 µg/kg feed) exposure individually and in combination (100 µg AFB1 + 1000 µg STC/kg feed) on the parameters of lipid peroxidation and glutathione redox system both in biochemical and gene expression levels in one-year-old common carp. Lipid peroxidation parameters were slightly affected, as significant differences were observed only in conjugated diene and triene concentrations. Reduced glutathione content decreased more markedly by STC than AFB1 or AFB1+STC, but glutathione peroxidase activity did not change. Expression of gpx4a, gpx4b, gss, and gsr genes was down-regulated due to STC compared to AFB1 or AFB1+STC, while an induction was found as effect of AFB1+STC in the case of gpx4a, but down-regulation for gpx4b as compared to AFB1. Expression of the glutathione biosynthesis regulatory gene, gss, was higher, but glutathione recycling enzyme encoding gene, gsr, was lower as an effect of AFB1+STC compared to AFB1. These results are supported by the changes in the expression of transcription factors encoding genes, nrf2, and keap1. The results revealed that individual effects of AFB1 and STC on different parameters are synergistic or antagonistic in multi-toxin treatment.

Single-Dose Toxicity of Individual and Combined Sterigmatocystin and 5-Methoxysterigmatocistin in Rat Lungs.

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are mycotoxins produced by common damp indoor Aspergilli series Versicolores. Since both STC and 5-M-STC were found in the dust of indoor occupational and living areas, their occupants may be exposed to these mycotoxins, primarily by inhalation. Thus, STC and 5-M-STC were intratracheally instilled in male Wistar rats using doses (0.3 mg STC/kg of lung weight (l.w.); 3.6 mg 5-M-STC/kg l.w.; toxin combination 0.3 + 3.6 mg/kg l.w.) that corresponded to concentrations detected in the dust of damp indoor areas in order to explore cytotoxicity, vascular permeability, immunomodulation and genotoxicity. Single mycotoxins and their combinations insignificantly altered lactate-dehydrogenase activity, albumin, interleukin-6, tumor necrosis factor-α and chemokine macrophage inflammatory protein-1α concentrations, as measured by ELISA in bronchioalveolar lavage fluid upon 24 h of treatment. In an alkaline comet assay, both mycotoxins provoked a similar intensity of DNA damage in rat lungs, while in a neutral comet assay, only 5-M-STC evoked significant DNA damage. Hence, naturally occurring concentrations of individual STC may induce DNA damage in rat lungs, in which single DNA strand breaks prevail, while 5-M-STC was more responsible for double-strand breaks. In both versions of the comet assay treatment with STC + 5-M-STC, less DNA damage intensity occurred compared to single mycotoxin treatment, suggesting an antagonistic genotoxic action.

Sterigmatocystin: Occurrence, toxicity and molecular mechanisms of action - A review.

The mycotoxin sterigmatocystin (STE) is produced mainly by Aspergillus fungi. It has been reported to occur in grains and grain-based products, cheese, coffee, spices and beer. The STE is a known biogenic precursor of aflatoxin B1, sharing with it several structural and biological similarities. The STE has been shown to be hepatotoxic and nephrotoxic in animals and it has been classified as possible human carcinogen (group 2B) by IARC. The STE has been reported to cause a marked decrease in cell proliferation in different mammalian cells. Data available on literature suggest that the cellular mechanisms underlying STE-induced toxicity include the induction of oxidative stress, mitochondrial dysfunction, apoptosis, cell cycle arrest, as well as alteration of immune system function and activation of different signalling pathways. Moreover, STE resulted to be genotoxic, being able to form DNA-adducts and induce DNA damage. Despite its strong cytotoxicity, no risk assessments have been still carried out by authorities due to the lack of toxicity data, so research on STE toxicological impact is still going on. This review reports information available regarding STE toxicity and its related mechanisms of action with the aim of updating information regarding last researches on this mycotoxin.

The role of mitochondria in sterigmatocystin-induced apoptosis on SH-SY5Y cells.

Mitochondria are cellular organelles involved in many crucial functions, such as generation of energy (ATP) and initiation of apoptosis. The aim of the present study was to evaluate the role of mitochondria in the toxicity induced by sterigmatocystin (STE), a mycotoxin produced by fungi of the genus Aspergillus, on SH-SY5Y cells. Our results showed that STE exposure decreased cell viability in a time- and concentration-dependent manner by MTT assay and caused mitochondrial dysfunction, as highlighted by the increase of STE cytotoxicity in cells forced to rely on mitochondrial oxidative phosphorylation. Furthermore, intracellular ATP depletion and increased mitochondrial reactive oxygen species were also observed. Since mitochondria play a pivotal role in apoptosis, the induction of this process in response to STE exposure was decided to study. Our results showed an increase in apoptotic cell population by flow cytometry, further confirmed by the up-regulation of the expression levels of the pro-apoptotic genes Bax and Casp-3 and the down-regulation of the anti-apoptotic gene Bcl-2 by qPCR technique. Taken together, our results provide novel insights in the signalling pathways of the cell death process induced by STE in SH-SY5Y cells, highlighting the key role played by mitochondria in STE toxicity.

Sterigmatocystin-induced cytotoxicity via oxidative stress induction in human neuroblastoma cells.

Sterigmatocystin (STE) is a mycotoxin produced by fungi of the genus Aspergillus. Considering that the effect of STE on neuronal system has not been well studied, the aim of the present study consists to investigate the cytotoxic effects of STE in human neuroblastoma (SH-SY5Y) cells. Moreover, the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation, lipid peroxidation (LPO) and antioxidant no-enzymatic (GSH) levels and enzymatic (GPx, GST, CAT and SOD) activity. Our results revealed that STE decreased cell viability in a dose and time-dependent manner. Furthermore, after 24 h of exposure, STE induced an increase in ROS generation and LPO at all concentrations tested (0.78, 1.56 and 3.12 µM), as well as a depletion of GSH levels, an increase in GSSG content and a decrease in GSH/GSSG ratio at the highest concentrations. The activity of all antioxidant enzymes resulted to be also decreased. Additionally, an enhance of the oxidative damage was caused by BSO, a GSH depletor, while NAC, a GSH precursor, showed a scavenger activity. Our findings suggest that STE could injure SH-SY5Y cells via oxidative stress and highlight the antioxidant role of the glutathione system.

Sterigmatocystin: A mycotoxin to be seriously considered.

Sterigmatocystin is a carcinogenic compound that affects several species of crops and several species of experimental animals. The sterigmatocystin biosynthetic pathway is the best known and most studied. The International Agency for Research on Cancer classifies sterigmatocystin in the Group 2B. Three groups of analytical methods to determine sterigmatocystin in food can be found: chromatographic, ELISA immunoassays and chemical sensors. In addition, sterigmatocystin is a precursor of aflatoxin B1 in those cases where cereals and/or food are contaminated with fungi capable of producing aflatoxins. Chemical structures of sterigmatocystin and aflatoxin B1 are similar. These mycotoxins are pathogens of animals and cereals, producing a major economic impact on biotechnology and agricultural and food industries. This review summarizes different aspects related to sterigmatocystin such as its biosynthesis, toxicological studies and analytical methods for its determination.

Genotoxicity of aflatoxins and their precursors in human cells.

Aflatoxins are found as food contaminant and some of them demonstrate a carcinogenic effect. The aflatoxins biosynthetic pathway involves 15 successive steps. The aim of this study was to compare the toxicity of aflatoxins and their precursors in three human cell lines. We tested the four aflatoxins and two of their metabolites; three early metabolic precursors and two late biosynthetic precursors. Cyclopiazonic acid, synthesized in parallel with aflatoxins, was also tested. The cytotoxicity and the genotoxicity was evaluated with the γH2AX assay in three human cell lines with different bioactivation capacities. Our results indicated that the most genotoxic chemicals in the three cell lines were in decreasing order sterigmatocystin (ST), aflatoxin B1 (AFB1), aflatoxicol (AFL), aflatoxin G1 (AFG1) and versicolorin A (VERA). Aflatoxin M1 (AFM1) demonstrated genotoxic property in only one cell line. The other tested compounds did not demonstrate any genotoxic activity. Overall, our results suggested different genotoxic mechanisms of action for the tested compounds, involving specific bioactivation pathways. Moreover, some metabolic precursors of aflatoxins demonstrated genotoxic potential equivalent or greater to AFB1. This should be taking into account for the development of new strategies intended to reduce the aflatoxins exposure and for human risk assessment.

Sterigmatocystin induced apoptosis in human pulmonary cells in vitro.

Sterigmatocystin (ST) is generally recognized as a potential carcinogen, mutagen and teratogen. Studies showed that ST could induce adenocarcinoma of lung in mice in vivo and DNA damage, cell cycle arrest in a human immortalized bronchial epithelial cell line (BEAS-2B cells) and a human lung cancer cell line (A549 cells) in vitro. Besides, ST could induce G2 arrest (cell cycle arrest in G2 phase) in several other cells. Cell cycle arrest may be one of the common toxic effects of ST. As cells may undergo apoptosis or death due to cell cycle arrest, we wondered whether apoptosis is another common effect of ST in different cells in vitro. In the present study, we studied the effects of ST on proliferation and apoptosis in A549 cells and BEAS-2B cells with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis (FCM). The MTT results showed that proliferation inhibition following ST treatment for 24h was observed in both A549 and BEAS-2B cells in vitro. And increased apoptosis by FCM was also found after ST treatment. Down-regulation of Bcl-2, up-regulation of Bax and the activation of caspase-3 after ST treatment were detected by western blotting analyses. The results in the present study are consistent with our previous results, which indicated that inducing apoptosis may be a common effect of ST in different cells in vitro.

Sterigmatocystin-induced checkpoint adaptation depends on Chk1 in immortalized human gastric epithelial cells in vitro.

Sterigmatocystin (ST) is a common contaminant detected in food and animal feed that has been recognized as a possible human carcinogen. Our previous studies demonstrate that ST causes DNA damage and subsequently triggers cell cycle arrest in G2 and apoptosis in immortalized human gastric epithelial cells (GES-1). Recently, studies have shown that in certain contexts, cells with DNA damage may escape checkpoint arrest and enter mitosis without repairing the damage. The term for this process is "checkpoint adaptation," and it increases the risk of unstable genome propagation, which may contribute to carcinogenesis. Thus, we aimed to investigate whether checkpoint adaptation occurs in GES-1 cells treated with ST and explored the underlying molecular mechanisms that contribute to this phenotype. In this study, we found that ST treatment for 24 h in GES-1 cells led to an initial G2 arrest; however, a fraction of GES-1 cells became large and rounded, and the number of p-H3-positive cells increased sharply after ST treatment for 48 h. Moreover, collection of the large and rounded cells by mechanical shake-off revealed that the majority of these large cells were found in the mitotic phase of the cell cycle. Importantly, we found that these rounded cells entered mitosis despite damaged DNA and that a small subset of this cell population survived and continued to propagate. These results suggest that ST induces an initial G2 arrest that is subsequently followed by G2 phase checkpoint adaptation, which may potentially promote genomic instability and result in tumorigenesis. Furthermore, we showed that activation of Chk1 contributes to the G2 arrest in GES-1 cells that are treated with ST for 24 h and that prolonged treatment of cells with ST for 48 h led to a decrease in the total protein and phosphorylation levels of Chk1 in mitotic cells, indicating that checkpoint adaptation may be driven by inactivation of Chk1. Knockdown studies confirmed that cells entered mitosis following inactivation of Chk1. Taken together, we show that ST treatment for 24 h activates Chk1 and induces a G2 arrest in GES-1 cells. However, prolonged ST treatment for 48 h led to Chk1 inactivation in GES-1 cells, which promotes checkpoint adaptation and entry of cells into mitosis despite damaged DNA. Importantly, checkpoint adaptation in GES-1 cells treated with ST may potentially promote genomic instability and drive tumorigenesis.

Sterigmatocystin induces G1 arrest in primary human esophageal epithelial cells but induces G2 arrest in immortalized cells: key mechanistic differences in these two models.

Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a "possible human carcinogen." Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM-Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53-p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53-p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM-Chk2 pathway, resulting in G1 phase arrest, whereas in SV40LT-immortalized human esophageal epithelial cells, SV40LT-mediated G1 checkpoint inactivation occurred, and ST-DNA damage activated p53-p21 signaling pathway, up-regulating G2/M phase regulatory proteins and finally leading to a G2 phase arrest. Thus, the SV40LT-mediated G1 checkpoint inactivation is responsible for the difference in the cell cycle arrest by ST between immortalized and primary cultured human esophageal epithelial cells.

Sterigmatocystin-induced oxidative DNA damage in human liver-derived cell line through lysosomal damage.

Sterigmatocystin (STC) is a carcinogenic and mutagenic mycotoxin produced by fungi of many Aspergillus species. As a precursor of the aflatoxins, STC is a risk factor of liver cancer. The objective of this study is to investigate STC-induced DNA damage and underlying mechanisms. The single cell gel electrophoresis (SCGE) assay was applied to assess DNA damage induced by STC. To clarify the underlying mechanisms, we measured the intracellular generation of reactive oxygen species (ROS) using dichlorofluorescein diacetate as a fluorochrome. The level of oxidative DNA damage was evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) and the acridine orange (AO) was used to measure the changes of lysosomal membrane stability. A significant dose-dependent increase of DNA strand breaks was found after treatment with STC (3 and 6 µM) respectively for 1 h. Increases in ROS level and the expression of 8-OHdG were also observed. A statistically significant increase in AO fluorescence intensity was observed in cells treated with STC (1.5, 3 and 6 µM) for 1 h. The DNA strand breaks induced by STC were almost prevented in cells pretreated with NH(4)Cl (10 mM) and NAC (10 mM) for 1 h. Our results thus indicated that STC exerts genotoxic effects on HepG2 cells, most likely through oxidative stress and lysosomal leakage.

Mycotoxins' activity at toxic and sub-toxic concentrations: differential cytotoxic and genotoxic effects of single and combined administration of sterigmatocystin, ochratoxin A and citrinin on the hepatocellular cancer cell line Hep3B.

Food safety organizations indicate the likelihood of constant human and animal exposure to mycotoxin mixtures as a possible negative public health impact. Risk assessment demonstrates that certain mycotoxins of Aspergillus and Penicillium spp. are toxic and hold a significant genotoxic efficacy at nanomolar concentrations. The aim of the current study was to investigate the potential cytogenetic effects of sterigmatocystin (STER), ochratoxin A (OTA) and citrinin (CTN) alone or in combination, at pM to µΜ concentrations, on the human hepatocellular cancer cell line Hep3B. MTT reduction, mitotic divisions, cell cycle delays and sister chromatid exchange rates (SCE) were determined as endpoints of metabolic activity, cytotoxicity, cytostaticity, and genotoxicity, respectively. All mycotoxin treatments induce SCE rates from 10-12 M, while their cytotoxic and cytostatic potential varies. In PRI and MI assays, but not at MTT, STER alone or in combination with OTA + CTN appeared cytostatic and cytotoxic, even at 10-12 M, while CTN alone and all other combinations displayed substantial cellular survival inhibition in doses ≥ 10-8 M. Co-administration of STER + OTA or STER + CTN in concentrations ≤ 10-1 M, increased the MI and MTT activity, while it did not affect the PRI. Mycotoxin co-treatments revealed in general similar-to-additive or antagonistic genotoxic and cytotoxic effects. Our results for the first time describe that STER alone or in combination with OTA and/or CTN share a cytotoxic and cytogenetic potential even at picoMolar concentrations on human hepatoma cells in vitro.

Metabolic detoxification pathways for 5-methoxy-sterigmatocystin in primary tracheal epithelial cells.

1. The health effects of inhaled mycotoxins remain poorly documented despite their presence in bioaerosols. 5-methoxy-sterigmatocystin is produced in association with sterigmatocystin by some Aspergillus spp., sometimes in larger amounts than sterigmatocystin. Whereas sterigmatocystin can be metabolized through cytochromes P450 (CYP), UDP-glucuronosyltransferases and sulfotransferases in airway epithelial cells, little is known about 5-methoxy-sterigmatocystin. 2. The 5-methoxy-sterigmatocystin metabolites were analyzed using human recombinant CYP and porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. The induction of xenobiotic-metabolizing enzymes was examined by real-time quantitative PCR for mRNA expression and 7-ethoxyresorufin O-deethylation activity. 3. CYP1A1 metabolized 5-methoxy-sterigmatocystin into hydroxy-nor-methoxy-sterigmatocystin, nor-methoxy-sterigmatocystin and dihydroxy-methoxy-sterigmatocystin. CYP1A2 led to monohydroxy-methoxy-sterigmatocystin. In PTEC, 5-methoxy-sterigmatocystin metabolism resulted into a glucuroconjugate of 5-methoxy-sterigmatocystin, a sulfoconjugate and a glucuroconjugate of monohydroxy-methoxy-sterigmatocystin. The exposure of PTEC for 24 h to 1 µM 5-methoxy-sterigmatocystin induced a significant increase in the mRNA levels of CYP1A1, without significant induction of the 7-ethoxyresorufin O-deethylation activity. 4. These data suggest that 5-methoxy-sterigmatocystin is mainly detoxified in airway cells through conjugation, as sterigmatocystin. However, while CYP produced a reactive metabolite of sterigmatocystin, no such metabolite was detected with 5-methoxy-sterigmatocystin. Nevertheless, 5-methoxy-sterigmatocystin increases the CYP1A1 mRNA levels. The long-term consequences remain unknown.

Effects of sterigmatocystin on esophageal epithelium and experimental reflux esophagitis in rats.

Patients with reflux esophagitis experience an increased incidence of esophageal cancer. In China, this may be the result of contamination of the food supply by Aspergillus fungi, which is known to harbor sterigmatocystin, a carcinogenic mycotoxin. To delineate the potential link between sterigmatocystin and esophageal cancer, an experimental model of reflux esophagitis was developed in rats that had undergone a cardiectomy and partial pylorus ligation. The rats were treated with sterigmatocystin or saline, and esophageal squamous cell hyperplasia was assessed based on the pathological evaluation. The expression of proliferating cell nuclear antigen (PCNA), transporter associated with antigen processing 1 (TAP1) and low molecular weight protein 2 (LMP2) was determined by immunohistochemistry. Intraperitoneal administration of sterigmatocystin promoted the proliferation of squamous epithelium. In addition, it also increased the expression of PCNA in esophageal epithelial cells in rats with reflux esophagitis and was correlated with the increased severity of epithelial hyperplasia. The expression levels of TAP1 and LMP2, which are located in the cytoplasm of esophageal epithelial cells, were reduced in rats with reflux esophagitis, and sterigmatocystin exposure further decreased the expression. Thus, the downregulation of TAP1 and LMP2 proteins by sterigmatocystin may directly affect tumor immunity by allowing transformed cells to escape the host immune surveillance, thereby promoting esophageal cancer.